Anti-glypican 3 antibody and pharmaceutical composition containing the same

ABSTRACT

The present invention relates to an antibody specifically binding to glypican 3 (GPC3), a nucleic acid encoding the antibody, a vector and a host cell containing the nucleic acid, a method of preparing the antibody, and a pharmaceutical composition for treating cancer or tumor, containing the antibody as an active ingredient. The antibody specifically binding to glypican 3 according to the present invention may be effectively used to treat cancer or tumor, particularly, hepatocellular carcinoma due to high affinity and specificity to glypican 3.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application of International Application No. PCT/KR2016/012193, filed Oct. 27, 2016, which claims the benefit of Korean Patent Application No. 10-2015-0150642 filed on Oct. 29, 2015, each of which is incorporated by reference in its entirety herein.

TECHNICAL FIELD

The present invention relates to an antibody specifically binding to glypican 3 (GPC3), a nucleic acid encoding the antibody, a vector and a host cell containing the nucleic acid, a method of preparing the antibody, and a pharmaceutical composition for treating cancer or tumor, containing the antibody as an active ingredient.

BACKGROUND ART

Glypican 3 was separated in the small intestine of a rat as a transcript in a developmental stage (Mol. Cell Biol. 8, 4243-4249, 1988), and thereafter, it was reported that a gene encoding glypican 3 was isolated from a glycosyl-phosphatidylinositol (GPI)-linked type sulfate human gastric cancer cell line having a core protein of a glypican family (molecular weight of 69 kDa) (Gene, 188, 151-156, 1997). It was reported that glypican 3 forms a protein-protein complex together with an insulin-like growth factor-2 and regulates actions of the insulin-like growth factor-2 (Nat. Genet., 12, 241-247, 1996). Particularly, it was known that glypican 3 is expressed in the fetal liver and placenta during development and in normal adult tissue, expression of glypican 3 is deteriorated or glypican 3 is not expressed at all.

Glypican 3 is known as a kind of oncofetal antigen that belongs to a glypican family of glycosyl-phosphatidylinositol (GPI)-anchored heparin sulfate proteoglycans, and cell membrane-bound glypican 3 is known to be composed of two subunits linked by one or more disulfide bonds.

In relation to functions and uses of glypican 3, it was reported that glypican may serve as a hepatocellular carcinoma marker, and it was suggested that glypican may serve as a receptor of endostatin having a possibility of an angiogenesis inhibitor, which was not clearly identified.

Recently, it has been known that glypican 3 is expressed in various cancers, particularly, hepatocellular carcinoma (HCC), melanoma, Wilm's tumor, and hepatoblastoma (Jakubovic and Jothy; Ex. Mol. Path. 82:184-189 (2007); Nakatsura and Nishimura, Biodrugs 19(2):71-77 (2005)), and it was reported that it is actually possible to treat tumors such as hepatic cancer, and the like, using an antibody against glypican 3 (Korean Patent No. 877,176, and the like).

Currently, several anti-glypican 3 antibodies were reported, but an antibody having a satisfactory therapeutic effect, particularly, an excellent cancer therapeutic effect has been not yet reported. Therefore, a need for an anti-glypican 3 antibody having a more excellent therapeutic effect is significant.

Therefore, the present inventors invented a novel antibody specifically binding to glypican 3 with high affinity, and confirmed a possibility of the antibody according to the present invention as an efficient anti-cancer agent, thereby completing the present invention.

DISCLOSURE

An object of the present invention is to provide a novel antibody specifically binding to glypican 3 (GPC3) with high affinity, a nucleic acid encoding the antibody, a vector and a host cell containing the nucleic acid, a method of preparing the antibody, and a pharmaceutical composition for treating cancer or tumor, containing the antibody as an active ingredient.

According to an aspect of the present invention, there is provided a novel antibody specifically binding to glypican 3 (GPC3) with high affinity.

According to another aspect of the present invention, there are provided a nucleic acid encoding an anti-glypican 3 antibody according to the present invention; a vector containing the nucleic acid; and a cell transduced with the vector, and a method of preparing an antibody according to the present invention, using the same.

According to another aspect of the present invention, there are provided a pharmaceutical composition for treating cancer or tumor, containing an anti-glypican 3 antibody according to the present invention as an active ingredient, and a method of treating cancer or tumor using the anti-glypican 3 antibody according to the present invention.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 is a view illustrating a result of confirming expression of GPC3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in each cell line.

FIG. 2 is a view illustrating a structure of a plasmid used for expression of GPC3 in each cell line.

FIG. 3 is a view illustrating that a cell line colony transformed so as to express GPC3 using resistance against Hygromycin may be confirmed.

FIG. 4 is a view illustrating a result of analyzing an expression amount of GPC3 in each cell line.

FIG. 5 is a view illustrating that an anti-glypican 3 antibody binds to a cell line transformed so as to express GPC3.

FIG. 6 is a view illustrating binding affinity of an anti-glypican 3 antibody according to the present invention to a transformed cell line (SK-Hep1-GPC3) so as to express GPC3.

FIG. 7 is a view illustrating binding affinity of the anti-glypican 3 antibody according to the present invention to a cell line (HepG2) expressing GPC3.

FIG. 8 is a view illustrating binding affinity of the anti-glypican 3 antibody according to the present invention to a cell line (HepG2) expressing GPC3.

FIG. 9 is a view illustrating results obtained by confirming whether or not GX102, GX270, and GS012 clone antibodies according to the present invention selectively bind to cell lines in proportion to an expression level of GPC3 in seven types of hepatic cancer cell lines (SK-Hep1, PLC/PRF/5, SNU398, Hep3B, Huh7, HepG2, and SK-Hep1-GPC3 #9) and one kind of normal hepatic cancer cell line (CHANG).

(a) SKHep1 (GPC3 negative cell line)

(b) SKHep1-GPC3 #9 (GPC3 positive cell line)

FIG. 10 is a view illustrating results obtained by confirming whether or not the GX102, GX270, and GS012 clone antibodies according to the present invention selectively bind to cell lines in proportion to an expression level of GPC3 in seven kinds of hepatic cancer cell lines (SK-Hep1, PLC/PRF/5, SNU398, Hep3B, Huh7, HepG2, and SK-Hep1-GPC3 #9) and one kind of normal hepatic cancer cell line (CHANG), representing and comparing the results by positive signs depending on an overlapping profile of fluorescence-activated cell sorting (FACS) histogram with that in an isotope negative control group.

DETAILED DESCRIPTION OF THE EMBODIMENTS

As used herein, the term “glypican 3” or “GPC3” collectively indicates arbitrary mutants, isomers, and homologues of glypican 3 as well as GPC3 present in animal, preferably, human bodies, as it is.

As used herein, the term “human GPC3” means GPC3 of a human and preferably has an amino acid sequence (SEQ ID NO: 393) of Genbank accession number AAH35972.1, but is not limited thereto.

The present invention provides an anti-glypican 3 antibody having a heavy chain variable region including a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 133, 143, 149, 159, 169, 174, 184, 199, 262, 268, 273, 281, 285, 291, 297, 308, 353, 363, 374, 380, or 386;

-   -   a heavy chain CDR2 comprising an amino acid sequence of SEQ ID         NO:134, 144, 150, 160, 170, 175, 185, 200, 231, 263, 266, 269,         274, 282, 286, 292, 298, 303, 309, 314, 318, 323, 328, 331, 336,         341, 345, 349, 354, 359, 364, 369, 375, 381, or 387; and     -   a heavy chain CDR3 comprising an amino acid sequence of SEQ ID         NO: 135, 139, 145, 151, 155, 161, 165, 171, 176, 180, 186, 189,         194, 195, 201, 205, 207, 210, 214, 218, 222, 227, 232, 237, 239,         243, 247, 250, 254, 258, 259, 264, 267, 270, 275, 278, 279, 283,         287, 293, 299, 304, 310, 315, 319, 324, 329, 332, 337, 342, 346,         350, 355, 360, 365, 370, 376, 382, or 388.

In another aspect, the present invention provides an anti-glypican 3 antibody having a light chain variable region including a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 136, 140, 146, 152, 156, 162, 166, 172, 177, 181, 187, 190, 193, 196, 202, 208, 211, 215, 219, 223, 224, 228, 233, 240, 244, 251, 255, 260, 265, 271, 288, 294, 300, 305, 311, 316, 320, 325, 333, 338, 343, 347, 356, 361, 366, 371, 377, 383, or 389;

-   -   a light chain CDR2 comprising an amino acid sequence of SEQ ID         NO: 137, 141, 147, 153, 157, 163, 167, 178, 182, 191, 197, 203,         209, 212, 216, 220, 225, 229, 234, 236, 241, 245, 252, 256, 276,         284, 289, 295, 301, 306, 312, 317, 321, 326, 330, 334, 339, 351,         357, 362, 367, 372, 378, 384, 390, or 392; and     -   a light chain CDR3 comprising an amino acid sequence of SEQ ID         NO: 138, 142, 148, 154, 158, 164, 168, 173, 179, 183, 188, 192,         198, 204, 206, 213, 217, 221, 226, 230, 235, 238, 242, 246, 248,         249, 253, 257, 261, 272, 277, 280, 290, 296, 302, 307, 313, 322,         327, 335, 340, 344, 348, 352, 358, 368, 373, 379, 385, or 391.

Preferably, the anti-glypican 3 antibody according to the present invention has a heavy chain variable region including a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 133, 143, 149, 159, 169, 174, 184, 199, 262, 268, 273, 281, 285, 291, 297, 308, 353, 363, 374, 380, or 386;

-   -   a heavy chain CDR2 comprising an amino acid sequence of SEQ ID         NO:134, 144, 150, 160, 170, 175, 185, 200, 231, 263, 266, 269,         274, 282, 286, 292, 298, 303, 309, 314, 318, 323, 328, 331, 336,         341, 345, 349, 354, 359, 364, 369, 375, 381, or 387; and     -   a heavy chain CDR3 comprising an amino acid sequence of SEQ ID         NO: 135, 139, 145, 151, 155, 161, 165, 171, 176, 180, 186, 189,         194, 195, 201, 205, 207, 210, 214, 218, 222, 227, 232, 237, 239,         243, 247, 250, 254, 258, 259, 264, 267, 270, 275, 278, 279, 283,         287, 293, 299, 304, 310, 315, 319, 324, 329, 332, 337, 342, 346,         350, 355, 360, 365, 370, 376, 382, or 388, and     -   a light chain variable region including a light chain CDR1         comprising an amino acid sequence of SEQ ID NO: 136, 140, 146,         152, 156, 162, 166, 172, 177, 181, 187, 190, 193, 196, 202, 208,         211, 215, 219, 223, 224, 228, 233, 240, 244, 251, 255, 260, 265,         271, 288, 294, 300, 305, 311, 316, 320, 325, 333, 338, 343, 347,         356, 361, 366, 371, 377, 383, or 389;     -   a light chain CDR2 comprising an amino acid sequence of SEQ ID         NO: 137, 141, 147, 153, 157, 163, 167, 178, 182, 191, 197, 203,         209, 212, 216, 220, 225, 229, 234, 236, 241, 245, 252, 256, 276,         284, 289, 295, 301, 306, 312, 317, 321, 326, 330, 334, 339, 351,         357, 362, 367, 372, 378, 384, 390, or 392; and     -   a light chain CDR3 comprising an amino acid sequence of SEQ ID         NO: 138, 142, 148, 154, 158, 164, 168, 173, 179, 183, 188, 192,         198, 204, 206, 213, 217, 221, 226, 230, 235, 238, 242, 246, 248,         249, 253, 257, 261, 272, 277, 280, 290, 296, 302, 307, 313, 322,         327, 335, 340, 344, 348, 352, 358, 368, 373, 379, 385, or 391.

In another aspect, the anti-glypican 3 antibody according to the present invention, contains a heavy chain variable region having a sequence with a sequence homology of 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more with an amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, or 131, or

-   -   contains a light chain variable region having a sequence with a         sequence homology of 80% or more, preferably 90% or more, more         preferably 95% or more, and most preferably 99% or more with an         amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,         18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,         50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,         82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,         110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132.

Preferably, the anti-glypican 3 antibody according to the present invention

-   -   contains a heavy chain variable region having a sequence with a         sequence homology of 80% or more, preferably 90% or more, more         preferably 95% or more, and most preferably 99% or more with an         amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,         19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,         51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,         83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,         111, 113, 115, 117, 119, 121, 123, 125, 127, 129, or 131, and     -   a light chain variable region having a sequence with a sequence         homology of 80% or more, preferably 90% or more, more preferably         95% or more, and most preferably 99% or more with an amino acid         sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,         24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,         56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,         88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,         116, 118, 120, 122, 124, 126, 128, 130, or 132.

Further, it is apparent to those skilled in the art that anti-glypican 3 antibodies of which some of the amino acids are substituted, inserted, and/or deleted in the heavy chain and light chain variable regions are also included in the scope of the present invention as long as characteristics such as an affinity and specificity to glypican 3, and the like, satisfying the object of the present invention are maintained. As an example, there is an antibody of which amino acid is conservatively substituted in a variable region. Conservative substitution means substitution with another amino acid residue having similar characteristics as those of an original amino acid sequence. For example, lysine, arginine, and histidine have similar characteristics in that they have basic side chains, and aspartic acid and glutamic acid have similar characteristics in that they have acidic side chains. Further, glycine, asparagines, glutamine, serine, threonine, tyrosine, cysteine, and tryptophane have similar characteristics in that they have uncharged polar side chains, alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar characteristics in that they have non-polar side chains, and tyrosine, phenylalanine, tryptophane, and histidine have similar characteristics in that they have aromatic side chains. Therefore, since it is apparent to those skilled in the art that even though an amino acid is substituted with another amino acid in an amino acid group having similar characteristics as described above, there is no particular change in characteristics, an antibody of which mutation is generated by conservative substitution in a variable region is also included in the scope of the present invention as long as the characteristics of the antibody according to the present invention are maintained.

As used herein, the term “antibody”, which is an immunoglobulin molecule immunologically reactive with a specific antigen, means a protein molecule serving as a receptor specifically recognizing antigen, and includes a whole antibody thereof and an antibody fragment thereof.

The antibody fragment in the present invention includes a short chain antibody, a diabody, a triabody, a tetrabody, a Fab fragment, a F(ab′)₂ fragment, Fd, scFv, a domain antibody, a minibody, a scab, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, derivatives of an antibody constant region, artificial antibodies based on protein scaffolds, and the like, which have binding affinity to glypican 3, but is not limited thereto. It is apparent to those skilled in the art that as long as binding affinity to glypican 3 is maintained, any type of antibody fragment according to the present invention exhibits the same characteristics as those of the antibody according to the present invention.

Meanwhile, in another aspect of the present invention, there is provide an antibody-drug conjugate (ADC) in which an anti-cancer drug having a tumor cell proliferation suppression effect is bound to the anti-glypican 3 antibody according to the present invention.

The drug capable of being used in the antibody-drug conjugate according to the present invention includes an arbitrary compound, moiety, or group having cytotoxicity or cell proliferation suppression effect, and includes (i) chemotherapeutic agents serving as a microtubulin inhibitor, a mitosis inhibitor, a topoisomerase inhibitor, or a DNA intercalator; (ii) a protein toxin performing an enzymatic function; and (iii) radioisotopes (radionuclides), and the like. One or more of the compounds may be used as the drug.

A non-restrictive example of the drug as described above may include maytansinoid, auristatin, dolastatin, trichothecene, CC1065, calicheamicin and other enediyne antibiotics, taxane, anthracycline, methotrexate, adriamycin, vindesine, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, daunomycin, etoposide, teniposide, carminomycin, aminopterin, dactinomycin, mitomycins, bleomycins, esperamicins, 5-fluorouracil, melphalan, other nitrogen mustards and stereoisomers, isosteres, analogues, or derivatives thereof, cis-platinum and cis-platinum analogues, enzymes and fragments thereof corresponding to other intercalating agents, for example, nucleolytic enzymes, antibiotics, and toxins (enzymatically active toxins or small molecule toxins of bacterial, fungal, plant or animal origin), various antitumor or anticancer agents such as cisplatin, CPT-11, doxorubicin, paclitaxel, docetaxel, and the like, but is not limited thereto. Further, an example of the radioisotope (radionuclide) includes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re, and the like, but is not limited thereto. A micro RNA (miRNA), a small interfering RNA (siRNA), and a small hairpin RNA (shRNA) capable of suppressing expression of a specific oncogene may also be used.

It is preferable that the anti-glypican 3 antibody provided in the present invention and the drug are conjugated to each other using a functional group such as a thiol group, or the like, of an amino acid residue such as lysine or cysteine, in the antibody. In this case, if necessary, the anti-glypican 3 antibody and the drug may also be conjugated to each other in a linker-mediated conjugate using a generally used linker. It is preferable to use a maleimide or iodoacetamide based linker. In the case of conjugating the drug to the antibody or the fragment thereof, it is preferable that the drug is conjugated to a C-terminal site opposite to an antigen binding site so as not to affect binding ability, specificity, and the like, of the antibody or the fragment thereof to glypican 3, and in the case of using the whole antibody instead of the fragment thereof, it is also preferable to conjugate the drug to an Fc region.

In addition, the present invention provides a chimeric antigen receptor (CAR)-based therapeutic agent containing the anti-glypican 3 antibody according to the present invention. For example, the therapeutic agent may be preferably a chimeric antigen receptor T-Cell or chimeric antigen receptor-natural killer (CAR-NK) cell therapeutic agent, but is not limited thereto.

In another aspect of the present invention, there is provided a bispecific antibody containing the anti-glypican 3 antibody according to the present invention. The bispecific antibody is an antibody capable of simultaneously binding to two kinds of antigens, and may be used in a general form in which a pair of different heavy chain and light chain capable of binding to antigens different from each other are linked, and may also be used in a form of a bispecific single chain antibody in which single-chain antibody fragments (scFv) in which VL and VH are linked to each other by a short linker peptide are linked in a form of scFv1-scFv2(-Fc), or a bispecific antibody using a BiTE technology by Micromet Inc. (Germany, see http://www.micromet.de).

The bispecific antibody according to the present invention may be preferably in a form in which the anti-glypican 3 antibody according to the present invention is bound to an antibody having binding ability to an immune effector cell-specific target molecule, or a fragment thereof. The immune effector cell-specific target molecule may be preferably selected from TCR/CD3, CD16(FcγRIIIa) CD44, Cd56, CD69, CD64(FcγRI), CD89, and CD11b/CD18(CR3), but is not limited thereto.

Further, the present invention provides a gene encoding the variable region of the anti-glypican 3 antibody according to the present invention and a recombinant vector containing the same. A polynucleotide, that is, a gene encoding light chain and heavy chain variable regions of the antibody according to the present invention or the fragment may be easily derived by those skilled in the art from an amino acid sequence of the anti-glypican 3 antibody provided in the present invention.

As used herein, the term “recombinant vector”, which is an expression vector capable of expressing a target protein in a suitable host cell, means a genetic construct containing an essential regulatory element to which a genetic insert is operably linked so as to be expressed. The gene encoding the anti-glypican 3 antibody according to the present invention may also be used in a form in which genes are inserted into separate vectors or one vector.

In detail, the polynucleotide encoding the amino acid sequence of the anti-glypican 3 antibody according to the present invention may also be used in a form in which genes are inserted into separate vectors, respectively or inserted into one vector, and may be used in a form in which polynucleotides encoding the heavy chain and light chain, the variable regions thereof, or the like, are inserted into separate vectors, respectively or inserted into one vector.

As used herein, the term “operably linked” means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding the target protein are functionally linked to each other so as to perform general functions. Operable linkage with a recombinant vector may be performed using a gene recombination technology wellknown in the art, and site-specific DNA cleavage and linkage may be easily performed using enzymes generally known in the art, or the like.

An expression vector suitable for producing the anti-glypican 3 antibody according to the present invention may include a signal sequence for membrane targeting or secretion in addition to expression regulatory elements such as a promoter, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer. The initiation codon and the termination codon may be generally considered as a portion of a nucleotide sequence encoding an immunogenic target protein, needs to be functional in an individual to whom a genetic construct has been administered, and must be in frame together with the coding sequence. The promoter may be generally constitutive or inducible. An example of the promoter available in prokaryotic cells may include lac, tac, T3, and T7 promoters, but is not limited thereto. An example of the promoter available in eukaryotic cells may include a β-actin promoter, promoters from human hemoglobin, human muscle creatine, and human metallothionein as well as a simian virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as a HIV Long Terminal Repeat (LTR) promoter, a moloney virus promoter, a cytomegalovirus (CMV) promoter, an epstein barr virus (EBV) promoter, a rous sarcoma virus (RSV) promoter, but is not limited thereto.

The expression vector may include a selectable marker for selecting host cells containing the vector. The selective marker is to select cells transformed by the vector, and as the selective marker, markers conferring selectable phenotypes such as drug resistance, nutrient requirement, cytotoxic agent resistance, or expression of surface proteins may be used. Since only cells expressing the selectable marker survive in the environment treated with a selective agent, transformed cells may be selected. Further, in the case of a replicable expression vector, the vector may include a replication origin, which is a specific nucleic acid sequence that initiates replication.

As the recombinant expression vector for inserting a foreign gene, a vector having various shapes such as plasmid, virus, cosmid, and the like, may be used. The kind of recombinant vector is not particularly limited as long as it may serve to express the desired gene and produce the desired protein in various host cells such as prokaryotic cells and eukaryotic cells, but a vector capable of mass-producing a foreign protein having a shape similar to that in a natural state while having a promoter having strong activity and having strong expression ability may be preferable.

Various expression host/vector combinations may be used to express the anti-glypican 3 antibody. An example of the expression vector suitable for eukaryotic cells includes an expression regulatory sequence derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus, but is not limited thereto. An example of the expression vector usable in bacteria host cells includes a bacterial plasmid obtained from Escherichia coli such as pET, pRSET, pBluescript, pGEX2T, a pUC vector, col E1, pCR1, pBR322, pMB9, and derivatives thereof; plasmids having a broader host range such as RP4; phage DNAs exemplified as various phage lambda derivatives such as λgt10, λgt11, and NM989; and other DNA phages such as M13, single-stranded filament type DNA phage, and the like. An expression vector available in yeast cells may be 2° C. plasmid and a derivative thereof. A vector useful for insect cells may be pVL941.

In another aspect, the present invention provides a host cell transformed with the recombinant vector. The recombinant vector is inserted in a host cell to form a transformant. A host cell suitable for the vector may be a prokaryotic cell such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis, or Staphylococcus sp. In addition, the suitable host cells may be eukaryotic cells, for example, fungal cells such as Aspergillus sp., yeast cells such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp., and Neurospora crassa, other lower eukaryotic cells, and cells of higher eukaryotes such as those from insects. In addition, the host cell may be derived from plants or mammals. Preferably, as the host cell, monkey kidney cells 7 (COS7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, Madin-Darby canine kidney (MDCK) cells, myeloma cell lines, HuT 78 cells, HEK293 cells, and the like, may be used, but the present invention is not limited thereto. Particularly, the CHO cells may be preferable.

As used herein, the term “transformation into host cells” may include any method of introducing nucleic acids into organisms, cells, tissues, or organs, and be performed by selecting standard techniques suitable for the host cell as known in the art. These methods may include an electroporation method, a protoplast fusion method, a calcium phosphate (CaPO₄) precipitation method, a calcium chloride (CaCl₂)) precipitation method, an agitation method using silicon carbide fiber, an agrobacterium-mediated transformation method, and a PEG, dextran sulfate, or lipofectamine and a desiccation/inhibition-mediated transformation method, but are not limited thereto.

In another aspect, the present invention provides a method of preparing the anti-glypican 3 antibody according to the present invention by culturing a host cell transformed with the recombinant vector.

A method of preparing a humanized antibody may include preparing a recombinant vector by inserting a nucleotide sequence encoding the anti-glypican 3 antibody according to the present invention into a vector; transforming a host cell with the recombinant vector to culture a transformant; and separating and purifying a humanized antibody from the cultured transformant.

In detail, the humanized antibody according to the present invention may be massproduced by culturing the transformant in which the recombinant vector is expressed in a nutrient medium. Here, general medium and culturing conditions may be suitably selected depending on the host cell. Conditions such as a temperature, pH of the medium, a culturing time, and the like, may be controlled appropriately for growth of the cells and mass production of proteins at the time of culturing the transformant.

The anti-glypican 3 antibody produced recombinantly as described above may be collected from the medium or cells lysates. In the case of a membrane-binding type anti-glypican 3 antibody, the anti-glypican 3 antibody may be isolated from a membrane by using a suitable surfactant solution (for example, triton-X 100) or enzymatic cleavage. Cells used for expressing the humanized antibody may be destructed by various physical or chemical means such as freezing-thawing acclimation, sonication, mechanical destruction, or a cell lysing agent, and be separated and purified by a general biochemical separation technology (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, Calif. (1990)). As the separation technology, electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immuno-adsorption chromatography, size exclusion chromatography, and the like), isoelectric focusing, and various modification and composite methods thereof, may be used, but the present invention is not limited thereto.

In another aspect, the present invention provides a composition for treating cancer, containing the anti-glypican 3 antibody. As used herein, the term “anti-cancer” includes “prevention” and “treatment”. Here, the term “prevention” means all actions for suppressing or delaying cancer diseases by administering the composition containing the antibody according to the present invention, and the term “treatment” means all actions for allowing symptoms of cancer to be alleviated or be advantageously changed by administering the antibody according to the present invention.

The kind of cancer or carcinoma capable of being treated by the composition according to the present invention is not particularly limited, and includes both solid cancer and blood cancer. Preferably, the cancer includes all kinds of cancer in which glypican 3 is expressed, and more preferably, the cancer may be selected from the group consisting of hepatic cancer, hepatocellular carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, larynx cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, uterine cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testis cancer, rectal cancer, head and neck cancer, cervical spinal cancer, ureteral cancer, osteosarcoma, neuroblastoma, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma and glioma. Most preferably, the cancer capable of being treated with the composition according to the present invention is hepatic cancer or hepatocellular carcinoma.

An anti-cancer composition according to the present invention may further contain a pharmaceutically acceptable carrier. In the case of a formulation for oral administration, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a flavoring agent, or the like, may be used, in the case of a formulation for injection, a buffering agent, a preservative, a soothing agent, a solubilizer, an isotonic agent, a stabilizer, or the like, may be mixed and used, and in the case of a formulation for local administration, a base, an excipient, a lubricant, a preservative, or the like, may be used. The formulation of the pharmaceutical composition according to the present invention may be variously prepared by mixing the pharmaceutical composition with the pharmaceutically acceptable carrier as described above. For example, in the case of the formulation for oral administration, the pharmaceutical composition may be prepared in forms of a tablet, a troche, a capsule, an elixir, a suspension, a syrup, a wafer, and the like, and in the case of the formulation for injection, the pharmaceutical composition may be prepared in a form of a unit-dose or multi-dose ampoule or vial. In addition, the anti-cancer composition may contain a surfactant for typically facilitating movement through a membrane. The surfactant as described above may be derived from steroids, or may be a cationic lipid such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), or the like, or various compounds such as cholesterol hemisuccinate, phosphatidyl glycerol, and the like.

In another aspect, the present invention provides a method of treating cancer and suppressing growth of cancer by administering the composition containing the anti-glypican 3 antibody according to the present invention to an individual. A pharmaceutically effective dose of the composition containing the anti-glypican 3 antibody according to the present invention may be administered in order to treat cancer cells or metastasis thereof, or suppress growth of cancer. The pharmaceutically effective dose may be changed by various factors such as the kind of cancer, an age, a weight, characteristics and degree of symptoms of a patient, the kind of current treatment method, a treatment frequency, an administration form and route, and the like, and may be easily determined by a specialist in the corresponding field. The composition according to the present invention may be administered together with the pharmacological or physiological ingredient, or sequentially administered. In addition, the composition according to the present invention and an additional therapeutic agent according to the related art may be combined with each other, and sequentially or simultaneously administered with each other. Administration as described above may be single dose administration or multi-dose administration. It is important to administer the composition at a minimum dose while obtaining a maximum effect without side effects in consideration of all the factors, which may be easily determined by those skilled in the art.

As used herein, the term “individual” means a mammal, preferably, a person in a state or with a disease, which may be alleviated, suppressed, or treated by administrating the humanized antibody or having the risk of the disease.

As used herein, the term “administration” means that a predetermined material is introduced to the individual by an appropriate method, and the composition containing the humanized antibody according to the present invention may be administered through any general route as long as a drug may arrive at a target tissue through the route. Examples of the administration include intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration, but are not limited thereto. However, since proteins are digested at the time of oral administration, it is preferable that the composition for oral administration is coated with an active drug or formulated so as to be protected from degradation in the stomach. In addition, the pharmaceutical composition may be administered by an optional device capable of moving an active material to target cells.

Hereinafter, the present invention will be described in detail through the Examples. However, these Examples are only to illustrate the present invention, and those skilled in the art will appreciate that these Examples are not to be construed as limiting a scope of the present invention.

Example 1: Preparation of Anti-Glypican 3 Antibody

1-1: Selection of Anti-Human GPC3 scFv Antibody Using Phage Display

An antibody selection process was performed using a phage display technology of inserting a DNA sequence to be expressed into a genome of a bacteriophage parasitic on E. coli using a genetic recombinant technology and expressing the inserted protein on a surface of the phage in a form in which the protein is fused with one of coating proteins of the phage. At the time of primary panning, 1 ml of library stock (10¹³ or more) was reacted in a GPC3-coated solid phase polystyrene tube (Nunc, 444202) at 37° C. for 2 hours. At the same time, 10 ul of XLI-Blue electroporation-competent cell (Stratagene) was inoculated into SB (10 ml)/tetracycline (100) and cultured so that OD600 was 0.8 to 1. The reactant at 37° C. for 2 hours was washed with 0.05% Tween 20/PBS (5 ml) four times, and from the secondary panning, as the number of panning was increased, the number of washing with 0.05% Tween 20/PBS (5 ml) was increased. Thereafter, the resultant was cultured in 1% BSA/0.1M Glycine (pH 2.0) at room temperature for 10 minutes, and phagemid was purified. The purified phagemid was transferred into a 50 ml tube and neutralized with 2M tris (70 ul). 9 ml of XLI-Blue Electroporation-Competent Cell (Stratagene) was treated, and 1 ml of XLI-Blue Electroporation-Competent Cell (Stratagene) was treated to the washed tube. After infection at room temperature for 30 minutes, SB (10 ml), tetracycline (20 μl), and carbenicillin (10 μl) were added thereto, and the infected cells were suspension-cultured at 37° C. and 220 rpm for 1 hour. Then, the cultured cells were treated with 1 ml of VCS M13 helper phage (1011 pfu), suspension-cultured at 37° C. and 220 rpm for 1 hour, treated with SB (80 ml), kanamycin (100 μl), and carbenicillin (100 μl), and cultured at 37° C. and 220 rpm for 12 hours or more. The cells cultured for 12 hours or more were centrifuged at 4° C. and 3500 rpm for 10 minutes, a supernatant was transferred to a new tube and 20% PEG/15% NaCl (20 ml) was added thereto and well-mixed, followed by reaction on ice for 30 minutes. Next, a supernatant was removed by centrifugation at 4° C. and 8000 rpm for 30 minutes, and the remaining pellet was collected and re-suspended in 1% BSA/PBS (2 ml), followed by centrifugation at 4° C. and 15000 rpm for 10 minutes. At this time, the collected pellet was removed and 1 ml of a total of 2 ml of supernatant was stored at −20° C., and 1 ml of the supernatant was used in the next panning.

1-2: Securing Individual Clone Using ELISA Method

A single colony from final amplification group of a phage display synthetic scFV library was collected, cultured in SB/carbenicillin (1.5 ml) at 37° C. and 220 rpm until OD 600 was 0.8 to 1.0 or so, and then cultured in 1 mM IPTG at 30° C. and 200 rpm for 12 hours or more. After the reactant was centrifuged at 5500 rpm for 5 minutes, only each of the supernatants was added to an ELISA plated with a GPC3 antigen, reacted at room temperature for 2 hours, and washed with PBST (1×PBS, 0.05% Tween 20) four times. Then, after a HRP/Anti-hFab-HRP conjugate diluted with 1% BSA/1×PBS at a ratio of 1/5000 was added thereto, reacted at room temperature for 1 hour, and washed with PBST (1×PBS, 0.05% Tween 20) four times again, a TMB solution was added thereto, and a reaction was carried out for 5 to 10 minutes. After adding the TMB stop solution thereto, an OD value was measured at a wavelength of 450 nm using a TECAN sunrise, a clone having a high OD value was secured as an individual clone.

As a result, 61 kinds of clones specifically binding to human GPC3 were selected, and amino acid sequences thereof were secured. The selected clones were named as clone GX090, clone GX092, clone GX099, clone GX102, clone GX107, clone GX114, clone GX116, clone GX118, clone GX119, clone GX122, clone GX184, clone GX186, clone GX189, clone GX196, clone GX197, clone GX201, clone GX205, clone GX206, clone GX207, clone GX209, clone GX213, clone GX214, clone GX216, clone GX217, clone GX219, clone GX221, clone GX222, clone GX224, clone GX225, clone GX226, clone GX229, clone GX233, clone GX234, clone GX235, clone GX242, clone GX245, clone GX247, clone GX248, clone GX253, clone GX259, clone GX263, clone GX264, clone GX265, clone GX268, clone GX270, clone GS001, clone GS002, clone GS003, clone GS004, clone GS005, clone GS006, clone GS007, clone GS008, clone GS009, clone GS010, clone GS011, clone GS012, clone GS013, clone GS014, clone GS015, clone GS016, clone GS017, clone GS018, clone GN328, clone GN337, and clone GN414, respectively. A variable region sequence of each of the clones was confirmed as illustrated in Table 1, and a CDR amino acid sequence in the variable region of each of the clones was confirmed as illustrated in Table 2 based on Kabat numbering.

TABLE 1 SEQ Variable ID Clone Region Amino Acid Sequencce NO: GX090 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQG 1 Chain LEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRS DDTAVYYCARGSSFSGFDPWGQGTLVTVSS Light QSVLTQPPSLSAAPGQKVTISCSGSSSNIGKNHVSWYQQFPGTA 2 Chain PKFLIYDNHRRPSGIPDRFSGSKSGTSATLDITGLQTGDEATYY CGTWDSSLSAVVFGGGTKLTVLG GX092 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQG 3 Chain LEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRS DDTAVYYCARADWGFFDYWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVTWYQQLPGTA 4 Chain PKLLIIYANNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYY CAAWDDSINGWVFGGGTKLTVLG GX099 Heavy EVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHMVROAFGQG 5 Chain LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRS EETAVYYCARYSSSPAKIDYWGQGTLVTVSS Light QSVLTQPPSVSAAPGEKVIISCSGSSSNIGKYYVSWYQQLPGTA 6 Chain PKLLIYDNNKRPSGIPDRFSGSKSGTSATLGISGLQTGDEADYY CGTWDSSLNLVFGTGTKVTVLG GX102 Heavy QMQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQG 7 Chain LEWMGWINPNSGGTNYAQKFOGRVTMTRDTSISTAYMELSRLRS DDTAVYYCARRGLRGDFDYWGQGTLVTVSS Light GQSVLTQPPSASETPGQKVTISCSGSSSNIGTNHVFWYQQLPGT 8 Chain APKLLIYRNNLRPSGVPDRFSGSKSGTSASLAISGLRSEDEADY YCAAWDDSLSWVFGGGTKLTVLG GX107 Heavy QMQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 9 Chain LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRS EDTAVYYCARSHGDYPEDYWGQGTLVTVSS Light YELTQPPSVSVSPGQTARIACSGDALPKHYAYWYQQKSGQAPVL 10 Chain VVYEDKKRPAGIPERFSGSSSGTVATLTISGAQVEDEAHYYCYS TDTSGNHRVFGGGTKLTVLG GX114 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 11 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVVDSSDYWGQGTLVTVSS Light AIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP 12 Chain KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLPLTFCGGTKVEIKR GX116 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 13 Chain LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRS EDTAVYYCARVMVRGVSTFGYWGQGTLVTVSS Light QSVLTQPPSVSAAPGQKVSISCSGSSSNIGKNFLSWYQQLPGTA 14 Chain PHLLIYRNNRRPSGIPDRFSGSKSGTSATLTITGLQTGDEGDYY CGAWDSRLSGVVFGGGTKLTVLG GX118 Heavy QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPG 15 Chain KGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVT AADTAVYYCARLRSFADNYVWGSYASYYFDYWGRGTLVTVSS Light QSVVTQPPSVSAAPGQRVTISCSGSSSNIGNNFVSWHQQLPGTA 16 Chain PKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYY CGTWDNSLGTGVFGGGTKLTVLG GX119 Heavy EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKG 17 Chain LEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARDRSWNYYGMDVWGQGTTVTVSS Light AIRMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP 18 Chain KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPVTFGGGTKLEIKR GX122 Heavy EVQLVESGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 19 Chain LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRS EDTAVYYCARDGGSSPDIWGQGTMVTVSS Light SYELTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKSGQAPV 20 Chain LVIYEDKKRPSEIPERFSGSSSGTMATLTITGAQVDDEADYYCY SIDRSGSRGVFGGGTKLTVLG GX184 Heavy QLQLQESGPGLVEPSETLSLTCAVSGGSISSSNNYWGWIRQPPG 21 Chain KGLEWIGSIFYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVT AADTAVYYCARLPTGTPGFYFDYWGQGTLVTVSS Light SYELTQPPSVSKGLRQTATLTCTGNSNNVGNQGAAWLQQHQGHP 22 Chain PKLLSYRNNNRPSGISERFSASRSGNTASLTITGLQPEDEADYY CSAWDSSLSAWVFGGGTKLTVLG GX186 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 23 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARLQGYWGRGTLVTVSS Light DIQMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQ 24 Chain KPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQYYSTPPTFGGGTKLEIKR GX189 Heavy QLQLQESGPGLVKPSETLSLTCAVSGGSISSSNNYWGWIRQPPG 25 Chain KGLEWIGSIFYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVT AADTAVYYCARLPTGTPGFYFDYWGQGTLVTVSS Light SYVLTQPPSVSKGLRQTATLTCTGNSSNVGNQGAAWLQQHQGHP 26 Chain PKLLSYRNNNRPSGISERFSASKSGNTASLTITGLRPEDEADYY CSAWDSSLSAWVFGGGTKLTVLG GX196 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 27 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRD EDTAVYYCARVTGDYWGRGTLVTVSS Light EIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQ 28 Chain KPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCOQYYSTPLIFGGGTKVEIKR GX197 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQG 29 Chain LEWMGWISAYNGNTNYAQKLQGRVIMTTDTSTSTAYMELRSLRS DDTAVYYCASQGSGWLDYWGQGTLVTVSS Light NFMLTQPHSVSESPGKTVTISCTGSGGNIASNYVQWFQQRPGSA 30 Chain PTTVIYDDVQRPSGVPNRFSGSIDSSSNSASLSISGLKTEDEAD YYCQSYDRTYRGVFGGGTKLTVLG GX201 Heavy QVQLVESGGGLVNPGGSLRLSCAASGFTFSNYGMNWVRQAPGKG 31 Chain LEWVSSISTRSGYIFYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARDRPNRSGMDVWGQGTTVTVSS Light QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGK 32 Chain APKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEGDY YCSSYTSSDTLVFGSGTKVTVLG GX205 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 33 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVLLDYWGQGTLVTVSS Light AIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKFGKAP 34 Chain KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLLLTFGGGTKLEIKR GX206 Heavy QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPG 35 Chain KGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVT AADTAVYYCARLGDYGGNGYYFDYWGQGTLVTVSS Light QSVLTQPPSVSAASGQKVTVSCSGSSSNIGKNGVSWHQQLPGTA 36 Chain PKLLIYENNRRPSEIPDRFSGSKSGTSATLAITGLQTGDEADYY CGTWDSSLNAGVFGTGTKVTVLG GX207 Heavy EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQG 37 Chain LEWMGWISAYNGNTNYAQKLQGRVTMITDTSTSTAYMELRSLRS DDTAVYYCAREGIAAAGYYYGMDVWGQGTTVTVSS Light QSALTQPPSVSGSPGQSVTISCTGTSSDVGGYNRVSWYQQPPGT 38 Chain APKLMIYEVSNRPSGVPDRFSGSKSGNTASLTISGLQAEDEADY YCSSYTSSTTYVFGTGTKVTVLG GX209 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 39 Chain LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRS EDTAVYYCARGYYYGMDVWGQGTTVTVSS Light QSVLTQPPSASVSPGQTASITCSGDKLGDKYVFWYRQKPGQSPV 40 Chain LVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQ TWDSSTEVFGTGTKVTVLG GX213 Heavy QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPG 41 Chain KGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVT AADTAVYYCARLFWQQLTFDYWGQGTLVTVSS Light QSVVTQPPSVSAAPGQRVSISCSGSSSNIGKNHVIWHQQFPGTA 42 Chain PKLLISENNKRPSGIPDRFSASKSGTSATLDITGLQTGDEADYY CGTWDNSLSAGVFGGGTKLTVLG GX214 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 43 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRV DDTAVYYCATLYDFWSQGTLVTVSS Light NIQMTQSPSSLSASVGDRVTITCQASQDITNFLNWHQQKPGKAP 44 Chain ELLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDVGTYYC QQYDNLPLTFGGGTKVEIKR GX216 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 45 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVVDSSDYWGQGTLVTVSS Light EIVLTQSPATLSLSPGESATLSCRASQSVSSYLAWYQQKPGQAP 46 Chain RLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPVALTFGGGTKLEIKR GX217 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 47 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVSGDPKDWGQGTLVTVSS Light DIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAP 48 Chain RLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPRELFGPGTKVDIKR GX219 Heavy QMQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQG 49 Chain LEWMGRINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRS DDTAVYYCARVARYCSSTSCRTGGMDVWGQGTTVTVSS Light QSVLTQPPSASGTPGQRVTISCSGRSSNIGSRYVYWYQQLTGTA 50 Chain PKLLIFRNDQRPSGVPDRFSASKSGTSASLAISGLRSEDEADYF CATWDDSLSAWVFGGGTKLTVLG GX221 Heavy QLQLQESGPGLVKPSETLSLTCAVSGGSISSSNNYWGWIRQPPG 51 Chain KGLEWIGSIFYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVT AADTAVYYCARLPTGTPGFYFDYWGQGTLVTVSS Light SYVLTQPPSVSKDLRQTATLTCTGNSSNVGNQGAAWVQQHQGHP 52 Chain PKLLSYRNNKRPSGISERLSASRSGNTASLTITGLQPEDEADYY CSAWDSSLSAWVFGGGTKLTVLG GX222 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 53 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVTLDYWGRGTLVTVSS Light AIRMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP 54 Chain KLLIYDASNLETGVPSRFSGSGSETDFTFTISSLQPEDIATYYC QQYDSLPTTFGPGTKVDIKR GX224 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 55 Chain LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRS EDTAVYYCARSRPRSSSFDYWGRGTLVTVSS Light NFMLTQPHSVSESPGKTVTISCTVSSGSIAKNYVHWYQRRPGSA 56 Chain PTPLIYEDNRRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAD YYCQSYDDSGDRFVFGTGTKVTVLG GX225 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 57 Chain LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRS EDTAVYYCARESGPSGGMDVWGQGTTVTVSS Light SYELTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKPGQAPV 58 Chain LVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCS SYTSSSTRVVFGGGTKLTVLG GX226 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 59 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNTLYLQMNSLRA EDTAVYYCARVTLDYWGRGTLVTVSS Light DIVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP 60 Chain KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLPSITFGQGTRLEIKR GX229 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 61 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARVLLDYWGQGTLVTVSS Light DIVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP 62 Chain KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLWLTFGGGTKLEIKR GX233 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 63 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVVDSSDYWGQGTLVTVSS Light AIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP 64 Chain KLLIYDASNLETGVPSRFSGSGSGTDFTLRISRVEAEDVGIYYC MEVRYWPYNFGQGTKLEIKR GX234 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQG 65 Chain LEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRS DDTAVYYCARVGLGDFWSGDYYYYYYGMDVWGQGTTVTVSS Light SSELTQDPAVSVALGQTVRITCQGDSLRSSYASWYHQKPGQAPV 66 Chain LVMYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCH SRDSTGSHPNWVFGGGTKLTVLG GX235 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 67 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVSGDPKDWGQGTLVTVSS Light AIQMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQ 68 Chain KPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLXAED VAVYYCQQYYSTPLTFGGGTKLEIKR GX242 Heavy QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 69 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVVAAREDYWGQGTLVTVSS Light EIVLTQSPATLSLSPGERATLSCRATQSVGSYLAWYQQKPGQAP 70 Chain RLLIYDAFNRATGIPDRFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPLTFGGGTKVEIKR GX245 Heavy QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 71 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVSDDSPYWGQGTLVTVSS Light VIWMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP 72 Chain KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLLALTFGGGTKLEIKR GX247 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG 73 Chain LEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRS EDTAVYYCARTRKGSGYSYGYGMDVWGQGTTVTVSS Light SYELTQPPSVSVSPGQTASITCSGDKLGNKYVSWYQQKPGQSPV 74 Chain LVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEAGYYCQ TWDSSVVFGGGTKLTVLG GX248 Heavy QMQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQG 75 Chain LEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRS EDTAVYYCARVRGTGPRGGTFDYWGQGTLVTVSS Light QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTA 76 Chain PKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYY CGTWDSSLSAWVFGGGTKLTVLG GX253 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 77 Chain LEWVSYISSSGTTIYYADSVKGRFTISRDNAKKSLYLQMNSLRA EDTAVYYCARVRDSSGFWGRGTLVTVSS Light QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTA 78 Chain PKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYY CGTWDSSLSAWVVGGGTKLTVLG GX259 Heavy QVQLVQSGAEVKKPGESLRISCKASGYKFTNYWIAWVRQMPGKG 79 Chain LEWMGIIYPGDSDTRYSPSLQGQVTISADKSITTAYLQWSSLRA SDTAMYYCATIGLREGRWGQGTLVTVSS Light AIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQYPGKAP 80 Chain KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNTLPFGQGTKLEIKR GX263 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKG 81 Chain LEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVTPYYYYGMDVWGQGTTVTVSS Light QSVVTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTA 82 Chain PKLLIYDSNKRPPGIPDRFSGSKSGTSATLGITGLQTGDEADYY CGAWDSSLSAVVFGGGTKLTVLG GX264 Heavy QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPG 83 Chain KGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVT AADTAVYYCARLRWYSYGYFDVRGGYYFDYWGRGTLVTVSS Light QSVLTQPPSVSAASGQKVTVSCSGSSSNIGKNGVSWHQQLPGTA 84 Chain PKLLIYENNRRPSEIPDRFSGSKSGTSATLAITGLQTGDEADYY CGTWDSSLNAGVFGTGTKVTVLG GX265 Heavy QVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKG 85 Chain LEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARVHLDYWGQGTLVTVSS Light DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQ 86 Chain KPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED VAVYYCQQYYSTPPTFGQGTKVEIKR GX268 Heavy EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKG 87 Chain LEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARDRSWNYYGMDVWGQGTTVTVSS Light NIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP 88 Chain KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPTFGQGTRLEIKR GX270 Heavy QVQLVQSGAEVKKPGASVKVSCKTSGYTFTSSYMHWVRQAPGQG 89 Chain LEWMGIINPNSRGTNYAQKFQGRVTMTSDTSISTAYMELSSLIS DDTAVYYCASSSGDYPDYWGQGTLVTVSS Light SYELTQPPSVSVSPGQTARIACSGDALPKHYAYWYQQKSGQAPV 90 Chain LVVYEDKKRPAGIPERFSGSSSGTVATLTISGAQVEDEAHYYCY STDTSGNHRVFGGGTKLTVLG GS001 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYSMSWVRQAPGKG 91 Chain LEWVSGISPSGGNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARALLGCKSAYCYYAMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNAVNWYQQLPGTA 92 Chain PKLLIYADSKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGSWDSSLSGYVFGGGTKLTVLG GS002 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYSMSWVRQAPGKG 93 Chain LEWVSSISPGGSSTYYADSVKGRFTISRDNSKNTLYLQMSSLRA EDTAVYYCARYRVWHMTTGDYYSNAMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNAVSWYQQLPGTA 94 Chain PKLLIYDDNKRPSGVLDRFSGSKSGTSASLAISGLRSEDEADYY CGTWDDSLSGYVFGGGTKLTVLG GS003 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYSMSWVRQAPGKG 95 Chain LEWVSVISPGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARYRVMRSPHNPYYSYGMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVNWYQQLPGTA 96 Chain PKLLIYSNSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGSWDASLSGYVFGGGTKLTVLG GS004 Heavy EVQLLESGGGLVQPGGSLRLSCAVSGFTFSNYSMSWVRQAPGKG 97 Chain LEWVSAISPGSSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARYRVWAHSASSSYSNAMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNDVSWYQQLPGTA 98 Chain PKLLIYSDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGSWDDSLNGYVFGGGTKLTVLG GS005 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKG 99 Chain LEWVSVISPDSSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARAAVRRYGPSPYYYYAMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNYVTWYQQLPGTA 100 Chain PKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CATWDYSLSGYVFGGGTKLTVLG GS006 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYSMSWVRQAPGKG 101 Chain LEWVSAISPGGGSKYYADSVRGRFTVSRDNSKNTLYLQMNSLRA EDTAVYYCARYRVQKSAKNVYSSNGMDVWGQGTLVTVSS Light QSVLTQPPPASGTPGQRVTISCSGSSSNIGNNAVNWYQQLPGTA 102 Chain PKLLIYYDSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGAWDSSLSAYVFGGGTKLTVLG GS007 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYSMSWVRQAPGKG 103 Chain LEWVSVISHDGRSKYYADSVKGRFAISRDNSKNTLYLQMNSLRA EDTAVYYCAKFRVWKRTNAHSYANAMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRATISCTGSSSNIGSNSVSWYQQLPGTA 104 Chain PKLLIYANSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGTWDASLSAYVFGGGTKLTVLG GS008 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKG 105 Chain LEWVSVISPDNGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARARVHCRRDQCYSYGMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNNVTWYQQLPGTA 106 Chain PKLLIYANSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGAWDSSLNGYVFGGGTKLTVLG GS009 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYSMSWVRLAPGKG 107 Chain LEWVSVISPSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRA EHTAVYYCARYRVKQMLNQRSYSNAMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNYVSWYQQLPGTA 108 Chain PKLLIYDDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGSWDASLSGYVFGGGTKLTVLG GS010 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYSMSWVRQAPGKG 109 Chain LEWVSVISPSSGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARYRVRQLKHTRSYADAMDVWGQGTLVTVSS Light QSVLTQPPSASGAPGQRVTISCSGSSSNIGSNAVSWYQQLPGTA 110 Chain PKLLIYANSHRPSGVPDRFSGSKSGTSASLAISGLRSEDETDYY CASWDSSLSGYVFGGGTKLTVLG GS011 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKG 111 Chain LEWVSGISPGGSSIYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARYAVYFLRSHGSYDYGMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNDVTWYQQLPGTA 112 Chain PKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGAWDYSLNAYVFGGGTKLTVLG GS012 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYSMSWVRQAPGKG 113 Chain LEWVSGISPGSGSKYYADSVRGRFTVSRDNSKNTLYLQMNSLRA EDTAVYYCARRARRFDYWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNAVTWYQQLPGTA 114 Chain PKLLIYSNSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGSWDSSLNGYVFGGGTKLTVLG GS013 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKG 115 Chain LEWVSSISSGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARKKSQFDYWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTA 116 Chain PKLLIYANSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CDTWDYSLSGYVFGGGTKLTVLG GS014 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKG 117 Chain LEWVSVISPNSGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCARPRILRRRVDHSYSYAMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNYVSWYQQLPGTA 118 Chain PKLLIYSNSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGTWDYSLSGYVLGGGTKLTVLG GS015 Heavy EVOLLESGGGLAQPGGSLRLSCAASGFTFSDYSMSWVRQAPGKG 119 Chain LEWVSAISPDGGSKYYADSVKGRFTISRDNSKNTLYLQWNSLRA EDTAVYYCARFRVIKLRAGWYSANGMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNDVSWYQQLPGTA 120 Chain PKLLIYYNSKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGAWDDSLNGYVFGGGTKLTVLG GS016 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKG 121 Chain LEWVSGIYSGNGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARALSSCPRGPCYYDDGMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVIWYQQLPGTA 122 Chain PKLLIYDNSKRPSGVPDRFSGSKSGTSASLXISGLRSGDEADYY CGTWDASLSAYVFGGGTKLTVLG GS017 Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYAMSWVRQAPGKG 123 Chain LEWVSGISPGDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARVARMCQGWRCSYADGMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNSVYWYQQLPGTA 124 Chain PKLLIYSDSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGTWDSSLSGYVFGGGTKLTVLG GS018 Heavy EVQLLESGGGLVQPGGSLRLSCAASGLTFSNYAMSWVRQAPGKG 125 Chain LEWVSVISPGSGSKYYADSVKGRFTVSRDNSKNTLYLQMNSLRA EDTAVYYCARHRVIKINRQTYYDYGMDVWGQGTLVTVSS Light QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVSWYQQLPGTA 126 Chain PKLLIYSDNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY CGAWDDSLSAYVFGGGTKLTVLG GN328 Heavy EVQLVESGGGLVQPGGSLRVSCAASGFTFSTAWMDWVRQAPGKG 127 Chain LEWVANINPDGSEKHYVDSVKGRFTVSRDNAKNSVYLHMNTLRA EDTAVYYCSKALDYWGQGTTVIVSS Light DIQMTQSPSTLSASVGDRVTITCRASQNINTWLAWYQQKPGKAP 128 Chain EVLIYEASSLESGVPPRFSGSGSGTEFTLTISSLQPDDSATYYC QQYNTYSPTFGQGTKLEIKR GN337 Heavy EVQLVESGPGLVKPSQTLSLTCTVSGGSVSSGGYHWNWIRQHPG 129 Chain KGLEWIGYIFNSGNTDYNPSLRSRLTISQDTSKNQFSLKLSSVT AADTAVYYCARHRSALLRWFDYWGQGTMVTVSSASTKGPSVFPL APSSKSTSG Light DVVLTQSPSSLSAFVGDRVTITCQASQDIRKYLNWFQQKPGKAP 130 Chain KLLIYDASTLETGVSSRFSGSGSGTHFNLVISSLEPEDSATYYC QQHDFVPRTFGQGTKLEIKR GN414 Heavy QVQLVQSGAEVKKPGASVKVSCKASGYTFSTYGITWVRQAPGQG 131 Chain LEWMGRITPNNGVTDYAQKFQGRVTMTRDTSISTAYMELSSLRS DDSAVYYCAREIGSSSWKLLDPWGQGTMVTVSS Light QAVLTQPSSVSVSPGQTASITCTGNSNNVGYEGAAWVQQYQGHP 132 Chain PKLLSDRNHNRPSGISERFSASRSGNTASLTITGLQSEDEADYY CSAWDSSLSEWVSGGGTKLTVLG

TABLE 2 Variable Clone Region CDR1 CDR2 CDR3 GX090 Heavy SYGIS WISAYNGNTNYAQKLQG GSSFSGFDP Chain (SEQ ID NO: (SEQ ID NO: 134) (SEQ ID NO: 135) 133) Light SGSSSNIGKNHVS DNHRRPS GTWDSSLSA Chain (SEQ ID NO: (SEQ ID NO: 137) (SEQ ID NO: 138) 136) GX092 Heavy SYGIS WISAYNGNTNYAQKLQG ADWGFFDY Chain (SEQ ID NO: (SEQ ID NO: 134) (SEQ ID NO: 139) 133) Light SGSSSNIGSNTVT ANNQRPS AAWDDSLNG Chain (SEQ ID NO: (SEQ ID NO: 141) (SEQ ID NO: 142) 140) GX099 Heavy SYYMH IINPSGGSTSYAQKFQG YSSSPAKIDY Chain (SEQ ID NO: (SEQ ID NO: 144) (SEQ ID NO: 145) 143) Light GSSSNIGKYYVS DNNKRPS GTWDSSLNA Chain (SEQ ID NO: (SEQ ID NO: 147) (SEQ ID NO: 148) 146) GX102 Heavy GYYMH WINPNSGGTNYAQKFQG RGLRGDFDY Chain (SEQ ID NO: (SEQ ID NO: 150) (SEQ ID NO: 151) 149) Light SGSSSNIGTNHVF RNNIRPS AAWDDSLS Chain (SEQ ID NO: (SEQ ID NO: 153) (SEQ ID NO: 154) 152) GX107 Heavy SYYMH IINPSGGSTSYAQKFQG SHGDYPEDY Chain (SEQ ID NO: (SEQ ID NO: 144) (SEQ ID NO: 155) 143) Light SGDALPKHYAY EDKKRPA YSTDTSGNH Chain (SEQ ID NO: (SEQ ID NO: 157) (SEQ ID NO: 158) 156) GX114 Heavy DYYMS YISSSGSTIYYADSVKG VVDSSDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 161) 159) Light QASQDISNYLN DASNLET QQYDNLP Chain (SEQ ID NO: (SEQ ID NO: 163) (SEQ ID NO: 164) 162) GX116 Heavy SYYMH IINPSGGSTSYAQKFQG VMVRGVSTFGY Chain (SEQ ID NO: (SEQ ID NO: 144) (SEQ ID NO: 165) 143) Light SGSSSNIGKNFLS RNNRRPS GAWDSRLSG Chain (SEQ ID NO: (SEQ ID NO: 167) (SEQ ID NO: 168) 166) GX118 Heavy SSSYYWG SIYYSGSTYYNPSLES LRSFADNYV Chain (SEQ ID NO: (SEQ ID NO: 170) (SEQ ID NO: 171) 169) Light SGSSSNIGNNFVS DNNERPS GTWDNSLGT Chain (SEQ ID (SEQ ID NO: 147) (SEQ ID NO: 173) NO: 172) GX119 Heavy SYGMH VIWYDGSNKYYADSVKG DRSWNYYGMDV Chain (SEQ ID NO: (SEQ ID NO: 175) (SEQ ID NO: 176) 174) Light RASQSISSYLN AASSLQS QQSYSTP Chain (SEQ ID NO: (SEQ ID NO: 178) (SEQ ID NO: 179) 177) GX122 Heavy SYYMH IINPSGGSTSYAQKFQG DGGSSPDI Chain (SEQ ID NO: (SEQ ID NO: 144) (SEQ ID NO: 180) 143) Light SGDALPKKYAY EDKKRPSEI YSIDRSGSR Chain (SEQ ID NO: (SEQ ID NO: 182) (SEQ ID NO: 183) 181) GX184 Heavy SSNNYWG SIFYSGSTYYNPSLKS LPTGTPGFYFDY Chain (SEQ ID NO: (SEQ ID NO: 185) (SEQ ID NO: 186) 184) Light TGNSNNVGNQGAA RNNNEPS SAWDSSLSA Chain (SEQ ID NO: (SEQ ID NO: 392) (SEQ ID NO: 188) 187) GX186 Heavy DYYMS YISSSGSTIYYADSVKG LQGY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 189) 159) Light KSSQSVLYSSNNKN WASTRES QQYYSTP Chain YLA (SEQ ID NO: (SEQ ID NO: 191) (SEQ ID NO: 192) 190) GX189 Heavy SSNNYWG SIFYSGSTYYNPSLKS LPTGTPGFYFDY Chain (SEQ ID NO: (SEQ ID NO: 185) (SEQ ID NO: 186) 184) Light TGNSSNVGNQGAA RNNNRPS SAWDSSLSA Chain (SEQ ID NO: (SEQ ID NO: 392) (SEQ ID NO: 188) 193) GX196 Heavy DYYMS YISSSGSTIYYADSVKG VTGDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 194) 159) Light KSSQSVLYSSNNKN WASTRES QQYYSTP Chain YLA (SEQ ID NO: (SEQ ID NO: 191) (SEQ ID NO: 192) 190) GX197 Heavy SYGIS WISAYNGNTNYAQKLQG QGSGWLDY Chain (SEQ ID NO: (SEQ ID NO: 134) (SEQ ID NO: 195) 133) Light TGSGGNIASNYVQ DDVQRPS QSYDRTYR Chain (SEQ ID NO: (SEQ ID NO: 197) (SEQ ID NO: 198) 196) GX201 Heavy NYGMN SISTRSGYIFYADSVKG DRPNRSGMDV Chain (SEQ ID NO: (SEQ ID NO: 200) (SEQ ID NO: 201) 199) Light TGTSSDVGGYNYVS DVSNRPS SSYTSSDT Chain (SEQ ID NO: (SEQ ID NO: 203) (SEQ ID NO: 204) 202) GX205 Heavy DYYMS YISSSGSTIYYADSVKG VLLDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 205) 159) Light QASQDISNYLN DASNLET QQYDNLLA Chain (SEQ ID NO: (SEQ ID NO: 163) (SEQ ID NO: 206) 162) GX206 Heavy SSSYYWG SIYYSGSTYYNPSLKS LGDYGGNGYYFDY Chain (SEQ ID NO: (SEQ ID NO: 170) (SEQ ID NO: 207) 169) Light SGSSSNIGKNGVS ENNRRPS GTWDSSLNA Chain (SEQ ID NO: (SEQ ID NO: 209) (SEQ ID NO: 148) 208) GX207 Heavy SYGIS WISAYNGNTNYAQKLQG EGIAAAGYYYGMDV Chain (SEQ ID NO: (SEQ ID NO: 134) (SEQ ID NO: 210) 133) Light TGTSSDVGGYNRVS EVSNRPS SSYTSSTTY Chain (SEQ ID NO: (SEQ ID NO: 212) (SEQ ID NO: 213) 211) GX209 Heavy SYYMH IINPSGGSTSYAQKFQG GYYYGMDV Chain (SEQ ID NO: (SEQ ID NO: 144) (SEQ ID NO: 214) 143) Light SGDKLGDKYVF QDNKRPS QTWDSST Chain (SEQ ID NO: (SEQ ID NO: 216) (SEQ ID NO: 217) 215) GX213 Heavy SSSYYWG SIYYSGSTYYNPSLKS LFWQQLTFDY Chain (SEQ ID NO: (SEQ ID NO: 170) (SEQ ID NO: 218) 169) Light SGSSSNIGKNHVI ENNKRPS GTWDNSLSA Chain (SEQ ID NO: (SEQ ID NO: 220) (SEQ ID NO: 221) 219) GX214 Heavy DYYMS YISSSGSTIYYADSVKG LYDF Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 222) 159) Light QASQDITNFLN DASNLET QQYDNLP Chain (SEQ ID NO: (SEQ ID NO: 163) (SEQ ID NO: 164) 223) GX216 Heavy DYYMS YISSSGSTIYYADSVKG VVDSSDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 161) 159) Light RASQSVSSYLA DASNRAT QQRSNWPPVA Chain (SEQ ID NO: (SEQ ID NO: 225) (SEQ ID NO: 226) 224) GX217 Heavy DYYMS YISSSGSTIYYADSVKG VSGDPKD Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 227) 159) Light RASQSVSSNLA GASTRAT QQYNNWPR Chain (SEQ ID NO: (SEQ ID NO: 229) (SEQ ID NO: 230) 228) GX219 Heavy GYYMH RINPNSGGTNYAQKFQG VARYCSSTSCRTGGMDV Chain (SEQ ID NO: (SEQ ID NO: 231) (SEQ ID NO: 232) 149) Light SGRSSNIGSRYVY RNDQRPS ATWDDSLSA Chain (SEQ ID NO: (SEQ ID NO: 234) (SEQ ID NO: 235) 233) GX221 Heavy SSNNYWG SIFYSGSTYYNPSLKS LPTGTPGFYFDY Chain (SEQ ID NO: (SEQ ID NO: 185) (SEQ ID NO: 186) 184) Light TGNSSNVGNQGAA RNNKRPS SAWDSSLSA Chain (SEQ ID NO: (SEQ ID NO: 236) (SEQ ID NO: 188) 193) GX222 Heavy DYYMS YISSSGSTIYYADSVKG VTLDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 237) 159) Light QASQDISNYLN DASNLET QQYDSLP Chain (SEQ ID NO: (SEQ ID NO: 163) (SEQ ID NO: 238) 162) GX224 Heavy SYYMH IINPSGGSTSYAQKFQG SRPRSSSFDY Chain (SEQ ID NO: (SEQ ID NO: 144) (SEQ ID NO: 239) 143) Light TVSSGSTAKNYVH EDNRRPS QSYDDSGDR Chain (SEQ ID NO: (SEQ ID NO: 241) (SEQ ID NO: 242) 240) GX225 Heavy SYYMH IINPSGGSTSYAQKFQG ESGPSGGMDV Chain (SEQ ID NO: (SEQ ID NO: 144) (SEQ ID NO: 243) 143) Light SGDALPKQYAY KDSERPS SSYTSSSTR Chain (SEQ ID NO: (SEQ ID NO: 245) (SEQ ID NO: 246) 244) GX226 Heavy DYYMS YISSSGSTIYYADSVKG VTLDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 237) 159) Light QASQDISNYLN DASNLET QQYDNLP Chain (SEQ ID NO: (SEQ ID NO: 163) (SEQ ID NO: 164) 162) GX229 Heavy DYYMS YISSSGSTIYYADSVKG VLLDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 247) 159) Light QASQDISNYLN DASNLET QQYDNLW Chain (SEQ ID NO: (SEQ ID NO: 163) (SEQ ID NO: 248) 162) GX233 Heavy DYYMS YISSSGSTIYYADSVKG VVDSSDY Chain (SEQ ID (SEQ ID NO: 160) (SEQ ID NO: 161) NO: 159) Light QASQDISNYLN DASNLET MEVRYWP Chain (SEQ ID NO: (SEQ ID NO: 163) (SEQ ID NO: 249) (162) GX234 Heavy SYGIS WISAYNGNTNYAQKLQG VGLGDFWSGDYYYYYYGMDV Chain (SEQ ID NO: (SEQ ID NO: 134) (SEQ ID NO: 250) 133) Light QGDSLRSSYAS GKNNRPS HSRDSTGSHPN Chain (SEQ ID NO: (SEQ ID NO: 252) (SEQ ID NO: 253) 251) GX235 Heavy DYYMS YISSSGSTIYYADSVKG VSGDPKD Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 227) 159) Light KSSQSVLYSSNNEN WASTRES QQYYSTP Chain YLA (SEQ ID NO: (SEQ ID NO: 191) (SEQ ID NO: 192) 190) GX242 Heavy DYYMS YISSSGSTIYYADSVKG VVAAREDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 254) 159) Light RATQSVGSYLA DAFNRAT QQRSNWP Chain (SEQ ID NO: (SEQ ID NO: 256) (SEQ ID NO: 257) 255) GX245 Heavy DYYMS YISSSGSTIYYADSVKG VSDDSPY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 258) 159) Light QASQDISNYLN DASNLET QQYDNLLA Chain (SEQ ID NO: (SEQ ID NO: 163) (SEQ ID NO: 206) 162) GX247 Heavy SYYMH IINPSGGSTSYAQKFQG TRKGSGYSYGYGMDV Chain (SEQ ID NO: (SEQ ID NO: 144) (SEQ ID NO: 259) 143) Light SGDKLGNKYVS QDNKRPS QTWDSS Chain (SEQ ID NO: (SEQ ID NO: 216) (SEQ ID NO: 261) 260) GX248 Heavy SYAIS GIIPIFGTANYAQKFQG VRGTGPRGGTFDY Chain (SEQ ID NO: (SEQ ID NO: 263) (SEQ ID NO: 264) 262) Light SGSSSNIGNNYVS DNNKRPS GTWDSSLSA Chain (SEQ ID NO: (SEQ ID NO: 147) (SEQ ID NO: 138) 265) GX253 Heavy DYYMS YISSSGTTIYYADSVKG VRDSSGF Chain (SEQ ID NO: (SEQ ID NO: 266) (SEQ ID NO: 267) 159) Light SGSSSNIGNNYVS DNNKRPS GTWDSSLSA Chain (SEQ ID NO: (SEQ ID NO: 147) (SEQ ID NO: 138) 265) GX259 Heavy NYWI IIYPGDSDTRYSPSLQG IGLREGR Chain (SEQ ID NO: (SEQ ID NO: 269) (SEQ ID NO: 270) 268) Light RASQGISNYLA DASNLET QQYDN Chain (SEQ ID NO: (SEQ ID NO: 163) (SEQ ID NO: 272) 271) GX263 Heavy SYSMN SISSSSSYIYYADSVKG VTPYYYYGMDV Chain (SEQ ID NO: (SEQ ID NO: 274) (SEQ ID NO: 275) 273) Light SGSSSNIGNNYVS DSNKRPP GAWDSSLSA Chain (SEQ ID NO: (SEQ ID NO: 276) (SEQ ID NO: 277) 265) GX264 Heavy SSSYYWG SIYYSGSTYYNPSLKS LRWYSYGYFDVRGGYYFDY Chain (SEQ ID NO: (SEQ ID NO: 170) (SEQ ID NO: 278) 169) Light SGSSSNIGKNGVS ENNRRPS GTWDSSLNA Chain (SEQ ID NO: (SEQ ID NO: 209) (SEQ ID NO: 148) 208) GX265 Heavy DYYMS YISSSGSTIYYADSVKG VHLDY Chain (SEQ ID NO: (SEQ ID NO: 160) (SEQ ID NO: 279) 159) Light KSSQSVLYSSNNKN WASTRES QQYYSTP Chain YLA (SEQ ID NO: (SEQ ID NO: 191) (SEQ ID NO: 192) 190) GX268 Heavy SYGMH VIWYDGSNKYYADSVKG DRSWNYYGMDV Chain (SEQ ID NO: (SEQ ID NO: 175) (SEQ ID NO: 176) 174) Light RASQSISSYLN AASSLQS QQSYST Chain (SEQ ID NO: (SEQ ID NO: 178) (SEQ ID NO: 280) 177) GX270 Heavy SSYMH IINPNSRGTNYAQKFQG SSGDYPDY Chain (SEQ ID NO: (SEQ ID NO: 282) (SEQ ID NO: 283) 281) Light SGDALPKHYAY YEDKKRPA YSTDTSGNH Chain (SEQ ID NO: (SEQ ID NO: 284) (SEQ ID NO: 158) 156) GS001 Heavy SYSMS GISPSGGNKYYADSVKG ALLGCKSAYCYYAMDV Chain (SEQ ID NO: (SEQ ID NO: 286) (SEQ ID NO: 287) 285) Light TGSSSNIGSNAVN ADSKRPS GSWDSSLSG Chain (SEQ ID NO: (SEQ ID NO: 289) (SEQ ID NO: 290) 288) GS002 Heavy GYSMS SISPGGSSTYYADSVKG YRVWHMTTGDYYSNAMDV Chain (SEQ ID NO: (SEQ ID NO: 292) (SEQ ID NO: 293) 291) Light TGSSSNIGSNAVS DDNKRPS GTWDDSLSG Chain (SEQ ID NO: (SEQ ID NO: 295) (SEQ ID NO: 296) 294) GS003 Heavy NYSMS VISPGSGSTYYADSVKG YRVMRSPHNPYYSYGMDV Chain (SEQ ID NO: (SEQ ID NO: 298) (SEQ ID NO: 299) 297) Light SGSSSNIGSNDVN SNSQRPS GSWDASLSG Chain (SEQ ID NO: (SEQ ID NO: 301) (SEQ ID NO: 302) 300) GS004 Heavy NYSMS AISPGSSNKYYADSVKG YRVWAHSASSSYSNAMDV Chain (SEQ ID NO: (SEQ ID NO: 303) (SEQ ID NO: 304) 297) Light SGSSSNIGNNDVS SDSQRPS GSWDDSLNG Chain (SEQ ID NO: (SEQ ID NO: 306) (SEQ ID NO: 307) 305) GS005 Heavy NYAMS VISPDSSSKYYADSVKG AAVRRYGPSPYYYYAMDV Chain (SEQ ID NO: (SEQ ID NO: 309) (SEQ ID NO: 310) 308) Light TGSSSNIGNNYVS ADSHRPS ATWDYSLSG Chain (SEQ ID NO: (SEQ ID NO: 312) (SEQ ID NO: 313) 311) GS006 Heavy GYSMS AISPGGGSKYYADSVRG YRVQKSAKNVYSSNGMDV Chain (SEQ ID NO: (SEQ ID NO: 314) (SEQ ID NO: 315) 291) Light SGSSSNIGNNAVN YDSNRPS GAWDSSLSA Chain (SEQ ID NO: (SEQ ID NO: 317) (SEQ ID NO: 277) 316) GS007 Heavy NYSMS VISHDGRSKYYADSVKG FRVWKRTNAHSYANAMDV Chain (SEQ ID NO: (SEQ ID NO: 318) (SEQ ID NO: 319) 297) Light TGSSSNIGSNSVS ANSNRPS GTWDASLSA Chain (SEQ ID NO: (SEQ ID NO: 321) (SEQ ID NO: 322) 320) GS008 Heavy NYAMS VISPDNGSTYYADSVKG ARVHCRRDQCYSYGMDV Chain (SEQ ID (SEQ ID NO: 323) (SEQ ID NO: 324) NO: 308) Light TGSSSNIGSNNVT ANSQRPS GAWDSSLNG Chain (SEQ ID NO: (SEQ ID NO: 326) (SEQ ID NO: 327) 325) GS009 Heavy NYSMS VISPSGGNTYYADSVKG YRVKQMLNQRSYSNAMDV Chain (SEQ ID NO: (SEQ ID NO: 328) (SEQ ID NO: 329) 297) Light TGSSSNIGNNYVS DDSQRPS GSWDASLSG Chain (SEQ ID NO: (SEQ ID NO: 330) (SEQ ID NO: 302) 311) GS010 Heavy NYSMS VISPSSGSIYYADSVKG YRVRQLKHTRSYADAMDV Chain (SEQ ID NO: (SEQ ID NO: 331) (SEQ ID NO: 332) 297) Light SGSSSNIGSNAVS ANSHRPS ASWDSSLSG Chain (SEQ ID NO: (SEQ ID NO: 334) (SEQ ID NO: 335) 333) GS011 Heavy NYAMS GISPGGSSIYYADSVKG YAVYFLRSHGSYDYGMDV Chain (SEQ ID NO: (SEQ ID NO: 336) (SEQ ID NO: 337) 308) Light TGSSSNIGSNDVT SNNQRPS GAWDYSLNA Chain (SEQ ID NO: (SEQ ID NO: 339) (SEQ ID NO: 340) 338) GS012 Heavy NYSMS GISPGSGSKYYADSVRG RARRFDY Chain (SEQ ID NO: (SEQ ID NO: 341) (SEQ ID NO: 342) 297) Light TGSSSNIGNNAVT SNSQRPS GSWDSSLNG Chain (SEQ ID NO: (SEQ ID NO: 301) (SEQ ID NO: 344) 343) GS013 Heavy NYAMS SISSGGGSTYYADSVKG KKSQFDY Chain (SEQ ID NO: (SEQ ID NO: 345) (SEQ ID NO: 346) 308) Light SGSSSNIGSNTVN ANSQRPS DTWDYSLSG Chain (SEQ ID NO: (SEQ ID NO: 326) (SEQ ID NO: 348) 347) GS014 Heavy NYAMS VISPNSGSNTYYADSVKG PRILRRRVDHSYSYAMDV Chain (SEQ ID NO: (SEQ ID NO: 349) (SEQ ID NO: 350) 308) Light TGSSSNIGNNYVS SNSHRPS GTWDYSLSG Chain (SEQ ID NO: (SEQ ID NO: 351) (SEQ ID NO: 352) 311) GS015 Heavy DYSMS AISPDGGSKYYADSVKGR FRVIKLRAGWYSANGMDV Chain (SEQ ID NO: (SEQ ID NO: 354) (SEQ ID NO: 355) 353) Light SGSSSNIGNNDVS YNSKRPS GAWDDSLNG Chain (SEQ ID NO: (SEQ ID NO: 357) (SEQ ID NO: 358) 356) GS016 Heavy NYAMS GIYSGNGNTYYADSVKG ALSSCPRGPCYYDDGMDV Chain (SEQ ID (SEQ ID NO: 359) (SEQ ID NO: 360) NO: 308) Light SGSSSNIGSNDVT DNSKRPS GTWDASLSA Chain (SEQ ID NO: (SEQ ID NO: 362) (SEQ ID NO: 322) 361) GS017 Heavy GYAM GISPGDGSTYYADSVKG VARMCQGWRCSYADGMDV Chain (SEQ ID NO: (SEQ ID NO: 364) (SEQ ID NO: 365) 363) Light TGSSSNIGNNSVY SDSHRPS GTWDSSLSG Chain (SEQ ID NO: (SEQ ID NO: 367) (SEQ ID NO: 368) 366) GS018 Heavy NYAMS VISPGSGSKYYADSVKG HRVIKINRQTYYDYGMDV Chain (SEQ ID NO: (SEQ ID NO: 369) (SEQ ID NO: 370) 308) Light SGSSSNIGSNTVS SDNNRPS GAWDDSLSA Chain (SEQ ID NO: (SEQ ID NO: 372) (SEQ ID NO: 373) 371) GN328 Heavy TAWMD NINPDGSEKHYVDSVKG ALDY Chain (SEQ ID NO: (SEQ ID NO: 375) (SEQ ID NO: 376) 374) Light RASQNINTWLA EASSLES QQYNTYS Chain (SEQ ID (SEQ ID NO: 378) (SEQ ID NO: 379) NO: 377) GN337 Heavy SGGYHWN YIFNSGNTDYNPSL HRSRLLRWFDY Chain (SEQ ID NO: (SEQ ID NO: 381) (SEQ ID NO: 382) 380) Light QASQDIRKYLN DASTLET QQHDFVP Chain (SEQ ID NO: (SEQ ID NO: 384) (SEQ ID NO: 385) 383) GN414 Heavy TYGIT RITPNNGVTDYAQKFQG EIGSSSWKLLDP Chain (SEQ ID NO: (SEQ ID NO: 387) (SEQ ID NO: 388) 386) Light TGNSNNVGYEGAA SDRNHNRPS SAWDSSLSE Chain (SEQ ID NO: (SEQ ID NO: 390) (SEQ ID NO: 391) 389)

1-3: Measurement of Quantitative Binding Capacity of Anti-GPC3 Antibody to Antigen

Quantitative binding capacity (affinity) of GX102 and GX270 clone antibodies, purified anti-GPC3 antibodies, to recombinant human GPC 3 was measured using a Biacore T-200(GE Healthcare, U.S.) biosensor. After immobilizing GPC3 (Cat. No. 2119-GP-050, R&D systems) purified from HEK293 cells onto a CM5 chip (GE Healthcare) to an Rmax of 200 using an amine-carboxyl reaction, the GX102 or GX270 antibody sequentially diluted in a HBS-EP buffer solution (10 mM HEPES, pH7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) was flowed at a flow rate of 30 μL/min in a concentration range of 0.078 nM to 5 nM for an association time of 120 seconds and a dissociation time of 1800 seconds. Dissociation of the antibody bound to GPC3 was induced by flowing 10 mM Glycine-HCl (pH1.5) at a flow rate of 30 μL/min for 30 seconds (Table 3). Affinity was obtained as kinetic rate constants (K_(on) and K_(off)) and an equivalent dissociation constant (K_(D)) using a Biacore T-200 evaluation software (Table 4).

TABLE 3 SPR Biacore T200 Chip CM5 Running Buffer HBS-EP pH 7.4 Flow rate 30 μl/Min Association/Dissociation time 120 Sec/600 Sec IgG Conc. 0.078~5 nM, ½ serial dilution Regeneration 10 mM Glycine-HCl pH 1.5, 30 Sec

TABLE 4 K_(on) K_(off) K_(D) GX102 9.1 × 10⁶ 8.4 × 10⁻⁵ 9.2 × 10⁻¹² GX270 2.4 × 10⁶ 1.7 × 10⁻⁴ 7.0 × 10⁻¹¹

Example 2: Fluorescence Activated Cell Sorter (FACS) Analysis on Binding of Anti-Glypican 3 Antibody to Cancer Cell Expressing GPC3

In order to evaluate whether the anti-GPC3 antibody derived from the synthetic library was selectively bound to cells expressing GPC3, an expression amount of GPC3 was measured in a cancer cell line, and binding of the antibody was confirmed using a FACS experiment. Table 5 illustrates antibody clones used for FACS screening for cancer cells expressing GPC3.

TABLE 5 Source Number Clones Synthetic Library 41 GS001, GS018, GN328, GN337, GN414, GX090, GX092, GX099, GX102, GX107, GX118, GX119, GX122, GX184, GX206, GX207, GX209, GX225, GX234, GX247, GX248, GX253, GX263, GX268, GX270

2-1: Preparation of Cell Line Expressing GPC3

An expression amount of GPC3 mRNA in cells was confirmed in 8 kinds of hepatic cancer cell lines (Huh-7, HepG2, Hep3B, SNU398, SNU475, SNU449, PLC/PRF/5, and SK-Hep1) using a real time-polymerase chain reaction (RT-PCR) method. After a TrypLE Express solution was added to the cell lines cultured in a 6-well plate to detach the cell lines, a total RNA was collected using a trizol solution. In order to amplify the GPC3 mRNA, a forward primer (5′ GGA CTT GGC CAC GTT CAT G 3′) and a reverse primer (5′ ACC TCA GCC ACA GTC AAC GG 3′) were used. As a comparison reference for quantitative comparison, a primer set (5′ CTT CGC TCT CTG CTC CTC CT 3′, 5′ CCA GTG GAC TCC ACG ACG TA 3′) for a GAPDH mRNA was used. After the total RNA obtained by separation using the trizol solution was quantified using an OD quantification method, 100 ng of cell RNA and 0.5 pM primer were put into a Maxim RT-PCR premix tube and a total volume was set to 20 μL using a nuclease-free distilled water, and a reaction was carried out at 45° C. for 30 minutes. Immediately, after an inactivation process was performed at 94° C. for 5 minutes, a polymerase chain reaction (PCR) was performed for 30 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute. In order to complete an unreacted reaction after the last cycle, the resultant was additionally kept at 72° C. for 3 minutes, and then, 5 μL of a 5-fold concentrated agarose electrophoresis sample buffer (1.25 mg/mL Bromophenol Blue, 1.25 mg/mL xylene cyanol, 30% glycerol, 25 mM Tris, pH7.6) was added thereto. After each of the analysis samples was put on 1.5% agarose gel and subjected to electrophoresis at 100 volts for 15 minutes, GPC3 and GPADH DNA fragments amplified by RT-PCR were examined at a UV wavelength (FIG. 1).

Sequences of primers used for amplification of cDNA (amplicon size 758 bp) encoding GPC3 were as follows.

Forward primer (sense primer): 5′GGA CTT GGC CAC GTT CAT G 3′ Reverse primer (anti-sense primer): 5′AAC TCA GCC ACA GTC AAC GG 3′

In addition, sequences of primers used for amplification of cDNA (amplicon size 379 bp) encoding GAPDH were as follows.

Forward primer (sense primer): 5′CTT CGC TCT CTG CTC CTC CT 3′ Reverse primer (anti-sense primer): 5′CCA GTG GAC TCC ACG ACG TA 3′

In SK-Hep1 hepatic cancer cells confirmed as a GPC3 negative cell line, a plasmid (pCMV/GPC3) having a GPC3 expression unit and Hygromycin resistant gene was delivered into cells using a jet-polyethyleneimine (PEI) transfection system (Polyplus, 101-40) (FIG. 2). After 48 hours, the cell culture solution was replaced with a culture medium containing Hygromycin B (200 μg/mL). While the culture medium was replaced every 3 days, 7 kinds of colonies having resistance against Hygromycin were obtained (FIG. 3).

2-2: Analysis of Expression Amount of GPC3 in Cell Line Expressing GPC3 and Tumor Cell Line

GPC3 existing on surfaces of cells was measured with respect to a cell line (SK-Hep1-GPC3) in which GPC3 was artificially expressed using a FACS experiment. After detaching cells to be analyzed, cultured in a culture dish by adding a TrypLE Express solution thereto, the detached cells were put into a 50 mL tube and centrifuged at room temperature and 2000 rpm for 3 minutes to remove a culture medium, followed by washing with PBS once. The resultant was suspended using a FACS buffer, transferred to a round bottom tube, and centrifuged at room temperature and 2000 rpm for 3 minutes. A supernatant was removed, and the resultant was suitably dissolved using the FACS buffer so as to have a concentration of 4×105 ea/mL. Then, as a FACS analysis antibody for GPC3, a mouse anti-GPC3 antibody (R&D systems) was used at 4° C., and as an isotope control group, a mouse IgG (1 μg, R&D systems) was used. After 1 hour, the resultant was washed with the FACS buffer two times, the anti-mouse IgG antibody and PE conjugated were added thereto at an amount of 5 μL per sample and bound thereto at 4° C. for 30 minutes. After collecting cells by centrifugation at 2000 rpm for 3 minutes, the cells were re-suspended by adding a fixation buffer (500 μL), and measured using a FACS calibur (FIG. 4).

As a result, it was confirmed that GPC3 was positive in 8 kinds of clones (#2, #3, #5, #6, #7, #8, #9, and # Pool), and in the SK-Hep1 cell line, a mother cell line, GPC3 was negative, as illustrated in FIG. 4.

2-3: Analysis of Selective Binding of Anti-GPC3 Antibody to Cell Line Expressing GPC3

Whether or not anti-GPC3 antibody selectively binds to a cell line (SK-Hep1-GPC3) in which GPC3 was artificially over-expressed was measured using a FACS experiment. After detaching SK-Hep1-GPC3 cells by adding a TrypLE Express solution thereto, the detached SK-Hep1-GPC3 cells were put into a 50 mL tube and centrifuged at room temperature and 2000 rpm for 3 minutes to remove a culture medium, followed by washing with PBS once. The resultant was suspended using a FACS buffer, transferred to a round bottom tube, and centrifuged at room temperature and 2000 rpm for 3 minutes. A supernatant was removed, and the resultant was suitably dissolved using the FACS buffer so as to have a concentration of 4×10⁵ ea/mL. Then, a candidate antibody (1 μg) was added thereto at 4° C., and in an isotope control group, a human IgG (1 μg, Sigma) was added instead of the candidate antibody. After 1 hour, the resultant was washed with the FACS buffer two times, a goat anti-human IgG antibody and FITC conjugated were added thereto at an amount of 1 μL per sample and bound thereto at 4° C. for 30 minutes. After collecting cells by centrifugation at 2000 rpm for 3 minutes, the cells were re-suspended by adding a fixation buffer (500 μL), and measured using a FACS calibur (FIG. 5).

In order to compare binding capacity between the analyzed candidate antibodies, a reference antibody was set in each analysis, and the binding capacity was compared based on mean fluorescent intensity (MFI) (FIG. 6). Since the SK-Hep1-GPC3 cells were cell lines in which GPC3 was artificially over-expressed, it is impossible to predicate that the SK-Hep1-GPC3 cells maintained an original GPC3 structure as it is. Therefore, whether or not the anti-GPC3 antibody was selectively bound to cells even in hepatic cancer cells (HepG2) in which GPC3 was originally expressed was compared and analyzed by the same method as described above (FIGS. 7 and 8). As a result, it was confirmed that the antibodies having excellent binding capacity in both of the two cell lines (SK-Hep1-GPC3 and HepG2) were GX102, GX270, and GS012 clone.

In order to confirm whether or not the GX102, GX270, and GS012 clone antibodies having excellent binding capacity to GPC3 selectively bind to cell lines in proportion to an expression level of GPC3 in tumor cells, FACS binding was measured in a total of seven kinds of hepatic cancer cell lines (SK-Hep1, PLC/PRF/5, SNU398, Hep3B, Huh7, HepG2, and SK-Hep1-GPC3 #9) and one kind of normal hepatic cancer cell line (CHANG) (FIG. 9). The results were represented and compared by positive signs depending on an overlapping profile of a FACS histogram with that in an isotope negative control group (Table 6 and FIG. 10).

As a result, GX102 and GX270 candidate antibodies were selectively bound to the hepatic cancer cell lines in proportion to the expression level of GPC3 as illustrated in Table 6 and FIGS. 9 and 10. On the contrary, it was confirmed that the GS012 antibody was non-specifically bound to all of the hepatic cancer cell lines regardless of the expression level of GPC3.

TABLE 6 GPC3Ab SK-Hep1 PLC/PRF/5 CHANG SNU398 Hep3B Huh7 HepG2 SK-Hep1-GPC3 #9 GPCSexp. − − − −/+ + + + + + + + GS012 + + + + + + + + + + + + + + + + + + + + + + GX102 − − − − + + + + + + + GX270 − − + − − + + + +

INDUSTRIAL APPLICABILITY

An antibody specifically binding to glypican 3 according to the present invention may be effectively used to treat cancer or tumor, particularly, hepatocellular carcinoma due to high affinity and specificity to glypican 3.

Although the present invention has been described in detail based on particular features thereof, it is obvious to those skilled in the art that these specific technologies are merely preferable embodiments and thus the scope of the present invention is not limited to the embodiments. Therefore, the substantial scope of the present invention is defined by the accompanying claims and equivalent thereof.

SEQUENCE LISTING FREE TEXT

Attached electronic file. 

The invention claimed is:
 1. An anti-glypican 3 antibody comprising a heavy chain variable region and a light chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 149, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 150, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 151, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 152, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 153, and a light chain CDR3 comprising the amino acid sequence of SEO ID NO: 154; a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 281, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 282, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 283, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 156, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 284, and a light chain CDR3 comprising the amino acid sequence of SEO ID NO: 158; or a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 297, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 341, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 342, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 343, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 301, and a light chain CDR3 comprising the amino acid sequence of SEO ID NO:
 344. 2. The anti-glypican 3 antibody according to claim 1, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, 89, or
 113. 3. The anti-glypican 3 antibody according to claim 1, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, 90, or
 114. 4. A conjugate comprising the anti-glypican 3 antibody according to claim 1 and a drug.
 5. The conjugate according to claim 4, wherein the drug is an agent for treating cancer.
 6. The conjugate according to claim 5, wherein the drug is at least one selected from the group consisting of a microtubulin inhibitor, a mitosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a protein toxin performing an enzymatic function, a radioisotope, miRNA, shRNA and siRNA.
 7. The conjugate according to claim 5, wherein the drug is at least one selected from the group consisting of maytansinoid, auristatin, dolastatin, trichothecene, CC1065, calicheamicin, taxane, anthracycline, methotrexate, adriamycin, vindesine, vinca alkaloids, mitomycin C, chlorambucil, daunorubicin, daunomycin, etoposide, teniposide, carminomycin, aminopterin, dactinomycin, mitomycins, bleomycins, esperamicins, 5-fluorouracil, melphalan, nucleolytic enzymes, antibiotics, toxins, cisplatin, CPT-11, doxorubicin, paclitaxel and docetaxel.
 8. A bispecific antibody comprising the anti-glypican 3 antibody according to claim
 1. 9. The bispecific antibody according to claim 8, comprising the anti-glypican 3 antibody and an antibody having binding ability to an immune effector cell-specific target molecule.
 10. The bispecific antibody according to claim 9, wherein the immune effector cell-specific target molecule is selected from the group consisting of TCR/CD3, CD16, CD44, CD56, CD69, CD64, CD89 and CD11b/CD18.
 11. A polynucleotide encoding the anti-glypican 3 antibody according to claim
 1. 12. A recombinant vector comprising the polynucleotide according to claim
 11. 13. A host cell transformed with the recombinant vector according to claim
 12. 14. The host cell according to claim 13, wherein the cell is selected from the group consisting of animal cell, plant cell, yeast, E. coli, and insect cell.
 15. The host cell according to claim 13, wherein the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, Madin-Darby canine kidney (MDCK) cells, myeloma cell lines, HuT 78 cells, HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis, Staphylococcus sp., Aspergillus sp., Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp., and Neurospora crassa.
 16. A method of preparing an anti-glypican 3 antibody comprising culturing a host cell according to claim
 13. 17. A composition comprising the anti-glypican 3 antibody according to claim
 1. 